The increasing incidence of breast cancer and the worldwide prevalence of the disease in nulliparous women led us to investigate the role played by hormonal and reproductive influences on breast cancer risk. For this purpose we tested the susceptibility of young virgin and of parous rats to develop 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary cancer. We confirmed that the period of highest susceptibility is encompassed between the initiation of ovarian function and the first pregnancy. DMBA given after the first pregnancy failed to induce mammary cancer, an effect mimicked in virgin rats by a 21-day treatment with the placental hormone chorionic gonadotropin (hCG). In addition, hCG also inhibited tumor progression. HCG acts on the ovary, stimulating the secretion of estrogen, progesterone, and inhibin. Under these hormonal stimuli the mammary gland's terminal end buds differentiate into Iobules with reduced cell proliferation, steroid hormone receptor content, and carcinogen binding, and increased efficiency of carcinogen adduct removal. Differentiation also involved the expression of differentiation markers, including the synthesis of inhibin, a secreted protein with tumor suppressor activity, and activation of the apoptotic genes TRPM2, ICE, p53, c-myc, WAF-1 /CIP-1, bcI-XS, and p53. Cluster analysis of mammary gland RNAs obtained during and after pregnancy or hCG treatment and that were hybridized to cDNA array membranes containing 5,800 rat genes revealed that four different patterns of gene clustering were expressed at different time points, allowing us to correlate the different stages of development of the mammary gland with specific gene expression profiles. We propose to elucidate whether the mechanisms through which hCG induces differentiation of the normal breast, preventing the initiation of cancer, differ from those activated for the inhibition of cancer progression, and whether the effects are specific for hCG or could be reproduced by ovarian steroid hormones or synthetic drugs. For accomplishing these goals we propose to: 1) Determine whether the protection of virgin rats from mammary cancer by treatment with hCG is the result of the induction of a genomic imprinting in the mammary epithelium that is specific for this hormone, or similar to that induced by steroid hormone treatment; 2) Determine whether hCG selectively inhibits mammary carcinogenesis through the induction of a stable pattern of gene transcription mediated by cytosine methylation, or whether its histone acetylating properties lead to the reactivation of tumor suppressor genes through pathways exclusively activated by hCG or common for ovarian steroid hormones, and 3) Determine whether the inhibition of mammary carcinogenesis by hCG occurs predominantly by activation of the p53 pathway through inhibition of methyltransferase or through histone acetylation. Knowledge acquired through these studies will solidify and broaden the potential of this model for developing novel strategies for the prevention and treatment of breast cancer based on physiological mechanisms of gene expression regulation.